The nucleus tegmentalis dorsalis lateralis (ntdl) is an integral part of the limbic midbrain area. We have previously presented evidence that this nucleus contains cholinergic cell bodies. Therefore, the cholinergic projection sites of the ntdl were examined in rats by a biochemical technique. The ntdl was destroyed unilaterally and brains were assayed after 14 days survival for changes in choline acetyltransferase activity in discrete brain areas. A bilateral projection appears to exist to the anteroventral and the posterior nucleus of the thalamus. An ipsilateral projection to the lateral portion of the medial thalamic nucleus and the ventral lateral geniculate body was found. The ntdl cholinergic projection appears to be a principal pathway that interrelates the thalamic area of the limbic forebrain with the dorsal tegmental area of the limbic midbrain region and therefore may play a functional role in emotional behavior.